Autophagy is a self-degradation process whereby cells can orderly clear defective organelles and recycle macromolecules as a nutrient source. Autophagy is elevated and essential for the tumorigenic growth of KRAS- mutant pancreatic ductal adenocarcinoma (PDAC), providing the rationale for clinical evaluation of the autophagy inhibitor hydroxychloroquine (HCQ) for PDAC. Disappointingly, when used as monotherapy or in combination with standard of care, HCQ has shown limited to no clinical efficacy for PDAC. We recently determined that the treatment of PDAC with inhibitors of the key KRAS effector pathway, the RAF-MEK-ERK mitogenic activated protein kinase (MAPK) cascade, unexpectedly caused further elevation of autophagy, rendering PDAC acutely dependent on this process, and hypersensitive to autophagy inhibition. We determined that ERK inhibition impaired other critical processes that then led to compensatory upregulation of autophagy. Our findings, together with essentially identical conclusions by another independent co-published study, have led to the initiation of clinical trials evaluating either MEK (trametinib, binimetinib) or ERK (LY3214996) inhibitor in combination with HCQ for metastatic KRAS-mutant PDAC. While early observations from compassionate use of this combination support a significant clinical impact, our preliminary studies support our premise that we can improve upon this therapy. We propose three aims to further advance autophagy inhibition as an anti-RAS therapeutic approach. First, we will determine if the ERK MAPK + HCQ combination will be similarly effective in KRAS/NRAS/BRAF- mutant CRC (Aim 1). HCQ is a lysosome inhibitor and consequently not selective for autophagy. We hypothesize that inhibitors of the ULK1/2 serine/threonine protein kinases, key initiators of starvation-induced autophagy, will act as more specific autophagy inhibitors. However, as with all protein kinase inhibitors, inhibitor-induced compensatory mechanisms will promote resistance to ULK inhibitor efficacy. Additionally, a comprehensive determination of ULK1/2 substrates remains to be completed. Thus, we will determine the direct and compensatory effects of ULK inhibition on the phosphoproteome and kinome to critically evaluate ULK inhibitors as autophagy inhibitors (Aim 2). Our Aim 3 studies are based on our application of a 2,500-gene druggable genome CRISPR/Cas9 genetic-loss-of-function screen to identify genes that modulate CQ anti-tumor activity. The identified hits that either enhance or reduce CQ growth inhibition activity represent candidate combinations or biomarkers for CQ resistance, respectively. We have identified mediators of the DNA damage response and cell cycle regulators as two major classes of resistance-promoting genes. We will mechanistically dissect these relationships and determine how inhibition of members of these pathways influences autophagic flux. In summary, our studies will enhance our understanding of autophagy regulation in cancer and aid in the development of novel combination therapies to target autophagy for the treatment of KRAS-mutant cancers.